We are developing a new technology for single molecule DNA sequencing based on the incorporation of dNTPs labeled on the g-phosphate with fluorescent dyes. The label is cleaved from the nucleotide along with pyrophosphate when the nucleotide is incorporated into DNA by a polymerase. The released label is sorted from intact g-dNTPs in a microfluidics system and is identified in real time by single molecule fluorescence detection optics. In this proposal, we develop a novel nucleotide chemistry to enable the sorting scheme and we use directed evolution methods to adapt polymerases for utilization of the g-dNTPs. Single molecule sequencing promises to reduce the cost of DNA sequencing by several orders of magnitude compared to current methods. PROPOSED COMMERCIAL APPLICATIONS: A genomic DNA sequencing technology that reduces cost and eliminates the need for cloning, physical mapping and electrophoresis would have great appeal to both the academic and industrial genomic research communities.